Chromium is a very important analyte for environmental monitoring, and developing biosensors for chromium is a long-standing analytical challenge. In this work, in vitro selection of RNAcleaving DNAzymes was carried out in the presence of Cr 3+ . The most active DNAzyme turned out to be the previously reported lanthanide-dependent Ce13d DNAzyme. While the Ce13d activity was ~150-fold lower with Cr 3+ compared to that with lanthanides, the activity of lanthanides and other competing metals was masked by using a phosphate buffer, leaving Cr 3+ the only metal that can activate Ce13d. With 100 µM Cr 3+ , the cleavage rate is 1.6 h -1 at pH 6.Using a molecular beacon design, Cr 3+ was measured with a detection limit of 70 nM, significantly lower than the U.S. Environmental Protection Agency (EPA) limit (11 μM). Cr(VI) was measured after its reduction by NaBH4 to Cr 3+ , and it can be sensed with a similar detection limit of 140 nM Cr(VI), lower than the EPA limit of 300 nM. This sensor was tested for chromium speciation analysis in a real sample, supporting its application for environmental monitoring. At the same time, it has enhanced our understanding on the interaction between chromium and DNA.3

Most RNA-cleaving DNAzymes require a metal ion to interact with the scissile phosphate for activity. Therefore, few unmodified DNAzymes work with thiophilic metals because of their low affinity for phosphate. Recently, an Ag-specific Ag10c DNAzyme was reported via in vitro selection. Herein, Ag10c is characterized to rationalize the role of the strongly thiophilic Ag. Systematic mutation studies indicate that Ag10c is a highly conserved DNAzyme and its Ag binding is unrelated to C-Ag-C interaction. Its activity is enhanced by increasing Na concentrations in buffer. At the same metal concentration, activity decreases in the following order: Li > Na > K. Ag10c binds one Na ion and two Ag ions for catalysis. The pH-rate profile has a slope of ∼1, indicating a single deprotonation step. Phosphorothioate substitution at the scissile phosphate suggests that Na interacts with the pro-R oxygen of the phosphate, and dimethyl sulfate footprinting indicates that the DNAzyme loop is a silver aptamer binding two Ag ions. Therefore, Ag exerts its function allosterically, while the scissile phosphate interacts with Na, Li, Na, or Mg. This work suggests the possibility of isolating thiophilic metal aptamers based on DNAzyme selection, and it also demonstrates a new Ag aptamer.

The interaction between chromium ions and DNA is of great interest in inorganic chemistry, toxicology, and analytical chemistry. Most previous studies focused on in situ reduction of Cr(VI), producing Cr 3+ for DNA binding. Recently, Cr 3+ was reported to activate the Ce13d DNAzyme for RNA cleavage. Herein, the Ce13d is used to study two types of Cr 3+ and DNA interactions. First, Cr 3+ binds to the DNA phosphate backbone weakly through reversible electrostatic interactions, which is weakened by adding competing inorganic phosphate. On the other hand, Cr 3+ coordinates with DNA nucleobases forming stable crosslinks that can survive denaturing gel electrophoresis condition. The binding of Cr 3+ to different nucleobases was further studied in terms of binding kinetic and affinity by exploiting FAM-labeled DNA homopolymers. Once binding takes place, the stable Cr 3+ /DNA complex cannot be dissociated by EDTA, attributable to the ultra-slow ligand exchange rate of Cr 3+ . The binding rate follows the order of G > C >T ≈ A. Finally, Cr 3+ gradually loses its DNA binding ability after storing at neutral or high pH, attributable hydrolysis. This hydrolysis can be reversed by lowering the pH.This work provides a deeper insight into the bioinorganic chemistry of Cr 3+ coordination with DNA, clarifies some inconsistency in the previous literature, and offers practically useful information for generating reproducible results.3

Functional DNA includes aptamers and DNAzymes, and metal ions are often importantf or achieving the chemical functions of such DNA. Biosensors based on functional DNA have mainly been tested in aqueous buffers. By introducing organic solvents with much lower dielectric constants, the interaction between metal ions and DNA can be significantly enhanced, and this might affect the performance of DNA-based biosensors. In this work, the effect of ethanol on the activity of the EtNa DNAzyme was studied for Ca 2 + detection. With 30 %e thanol, the sensor has ad etection limit of 1.4 mm Ca 2 + ,w hich is a1 6-fold improvement relative to that in water.T his EtNa DNAzyme is unique because other tested DNAzymes are all inhibited by 50 %e thanol. Finally,b yu sing the EtNa DNAzyme as as caffold, the adenosinem onophosphate (AMP) aptamer was inserted to construct an aptazyme, which allowed the measurement of AMP in ethanol. In summary,t his study has reported the most sensitiveD NA-based sensorf or Ca 2 + ,a nd its sensitivity and selectivity can approach those of proteins or small-molecule ligands. Thisw ork also provides aw ay to measurea ptamer binding in organic solvents.In the past three decades, the functionofDNA has significantly expanded. In particular,D NA has emerged as ap owerful platform for biosensor development. [1][2][3][4][5] In addition to complementaryn ucleic acids, DNA can also detect variousm etal ions, small molecules, proteins, and even cells by using DNA aptamers and DNAzymes. The majority of detections, to date, have been performed in aqueous buffers, and an interesting question is whether it is possible to use DNA sensors in organic solvents. We reason such studies have at least the following implications and applications.F irst, it might broaden the range of analytes that can be detected, and enable such sensors in more research areas. In addition, for certain analytes,t he sensitivity might be even higher in solvents than that in water due to lower dielectric constants of organic solvents, or differences in other physicalproperties. Finally,itm ight allow new physical insights into DNA.DNA can maintain its base pairing in many water-miscible solvents up to ac ertain solvent fraction. [6,7] The most extensively studied solvent for DNA is ethanol. TheD NA duplex is first destabilizedw ith increasing ethanolc oncentration (e.g., lowered DNA melting temperature, T m ). [8] At ac riticale thanol concentration, which is af unctiono ft he type and concentration of cation in the system,D NA starts to aggregate. With 70-80 %e thanol, aB -to-A form transition occurs that is attributable to extensive DNA dehydration.M ost previouss tudies focusedo nt he stabilitya nd hybridization kinetics of DNA in organic solvents. [9][10][11][12][13] Since 1994, research on DNA catalysis has been explored. [14] DNAzymes are DNA-based catalysts, and they often require metal ions as cofactors for activity. [15][16][17][18] All known DNAzymes were obtained by ac ombinatorial biology technique called in vitro selection and D...

The EtNa DNAzyme was isolated during the isopropanol precipitation step of an in vitro selection effort. Although inactive with the intended cofactor, its RNA cleavage activity was observed under a few conditions. With Na , EtNa was highly active in ∼50 % ethanol, whereas in water, it was highly active with Ca . In this work, we showed that the EtNa DNAzyme was accelerated by freezing in water in the presence of Na . The apparent K value reached 6.2 mm Na under the frozen condition, over 20 times tighter than that in water at room temperature. With 10 mm Na , EtNa had a cleavage rate of 0.12 h after freezing at -20 °C. This effect was unique to EtNa, as all other tested DNAzymes were inhibited by freezing except for the Na -specific NaA43. Freezing also inhibited EtNa if Ca was used. We attributed this to the concentrations of EtNa and Na in the micropockets between ice crystals, but divalent metals might misfold DNA. Overall, we have systematically studied the effect of freezing on the RNA-cleavage activity of DNAzymes. The DNAzyme sequence and the metal ion species are both crucial to determine the effect of freezing.